Non-thermal isochoric freezing and brine retention: Quality and microbial safety of ready-to-eat hard-boiled eggs

Ready-to-eat hard-boiled eggs (HBEs) are prone to microbial contamination during post-processing and prolonged storage, posing potential food safety risks. This study evaluated the effects of in-package isochoric freezing (ICF) at -10 °C/89 MPa for 6 days on HBEs packaged in 0.3% salt solution (brine) and its impact on physicochemical quality during subsequent refrigerated storage for an additional 50 days at 4 °C, either with the brine retained in the pouch (ICF-w) or with the brine removed after 6 days of treatment (ICF-wo). Refrigerated storage (RF) under the same conditions (at 4 °C), with brine retained (RF-w) or removed after 6 days of storage (RF-wo), was included as a control. A subset of eggs was inoculated with Listeria innocua prior to ICF treatment to assess microbial reduction. Physical, biochemical, and microbial properties were assessed at 10-day intervals. Microbial counts, including Total Plate Count (TPC) and Yeast and Mold (Y&M), in ICF-w samples remained below the detection limit throughout storage, while the ICF treatment achieved a significant L. innocua reduction of 5.1-log₁₀ (p < 0.05). In contrast, microbial growth became detectable from day 16 and day 26 in RF-wo and ICF-wo samples, reaching 6.1-log₁₀ and 7.3-log₁₀ CFU/g by day 56, respectively. Both ICF-w and ICF-wo treatments showed significantly lower hardness values than their RF counterparts with reductions ranging from13.7% to 66.5% (p < 0.05). However, ICF-w HBEs maintained greater overall physicochemical quality stability, with lower weight loss (6.8% vs. 9.8% in ICF-wo samples on day 56), better retention of texture, and color, along with lower lipid oxidation, relative to ICF-wo samples. Overall, ICF-w treatment maintained microbiological stability throughout the 56-day storage period; however, progressive changes in weight loss and texture by the final storage point limited the optimal physicochemical quality shelf life to 46 days, highlighting the potential of ICF as a non-thermal preservation strategy.